A brief overview of a mouse model of cerebral hypoperfusion by bilateral carotid artery stenosis

Abstract

Vascular cognitive impairment (VCI) refers to all forms of cognitive disorder related to cerebrovascular diseases, including vascular mild cognitive impairment, post-stroke dementia, multi-infarct dementia, subcortical ischemic vascular dementia (SIVD), and mixed dementia. Among the causes of VCI, more attention has been paid to SIVD because the causative cerebral small vessel pathologies are frequently observed in elderly people and because the gradual progression of cognitive decline often mimics Alzheimer’s disease. In most cases, small vessel diseases are accompanied by cerebral hypoperfusion. In mice, prolonged cerebral hypoperfusion is induced by bilateral carotid artery stenosis (BCAS) with surgically implanted metal micro-coils. This cerebral hypoperfusion BCAS model was proposed as a SIVD mouse model in 2004, and the spreading use of this mouse SIVD model has provided novel data regarding cognitive dysfunction and histological/genetic changes by cerebral hypoperfusion. Oxidative stress, microvascular injury, excitotoxicity, blood-brain barrier dysfunction, and secondary inflammation may be the main mechanisms of brain damage due to prolonged cerebral hypoperfusion, and some potential therapeutic targets for SIVD have been proposed by using transgenic mice or clinically used drugs in BCAS studies. This review article overviews findings from the studies that used this hypoperfused-SIVD mouse model, which were published between 2004 and 2021.

Introduction

Vascular cognitive impairment (VCI) includes any types and stages of vascular-related cognitive decline from vascular mild cognitive impairment (VaMCI) to vascular dementia (VaD).^1,2 Among the causes of VCI, more attention has been paid to subcortical small vessel disease (SSVD) as frequently shown lesions in elderly patients with cognitive decline. ³ Early intervention for SSVD may be crucial for the reduction of the demented population. ⁴ Although the mechanisms and clinical manifestations have been reported, better diagnostic markers and satisfactory therapeutic options for SSVD are still under investigation. The most common pathology in SSVD is diffuse damage to the white matter. Dementia caused by SSVD has been known as subcortical ischemic vascular dementia (SIVD). ⁵ The management of VCI includes blood pressure control and checking lifestyle factors such as nonsmoking and physical activity for the prevention of cerebrovascular diseases. ⁶ In addition, cholinesterase inhibitors and N-methyl D-aspartate antagonist memantine showed mild positive effects on cognition, but data are insufficient to support widespread use of these drugs in vascular dementia. ⁷ Overall, disease-modifying treatments are not yet available partly because the epidemiology is not well understood and the cognitive decline is often heterogeneous (i.e., mixed pathology with Alzheimer’s disease or α-synucleinopathy). Further, evidence of intervention for lowering the risk of incident dementia or cognitive decline is also lacking. Despite advanced neuroimaging methods in humans, understanding of the pathophysiological mechanisms of cerebral small vessel disease is incomplete,^8–10 and the absence of circulating or cerebrospinal fluid biomarkers specific for VCI makes it challenging to monitor the efficacy of the interventions. Thus, for our deeper understanding of the pathological mechanisms of VCI and for finding a better therapeutic approach for this disease, more data are needed regarding the changes in cognitive behavior, histology, and gene and protein expression from rodent model studies.

In 1994, a rat model of cerebral hypoperfusion induced by occlusion of the bilateral common carotid arteries was developed for investigating chronic ischemic white matter lesions. ¹¹ Although this rat model showed glial activation and white matter changes resembling white matter lesions in humans, there were two potential limitations; (a) the visual pathway was damaged by the occlusion of the ophthalmic arteries which can affect behavioral tests, and (b) lack of knock-out or transgenic rats to conduct genetic studies. To overcome these points, in 2004, a mouse model of prolonged cerebral hypoperfusion by bilateral carotid artery stenosis (BCAS) was proposed as a more reasonable mouse model to study chronic ischemic white matter lesions. ¹² The BCAS mice do not show severe damage to the optic tract, and genetic studies can be performed with this model using knock-out/knock-in or transgenic mice. The authors, who developed this mouse model, reported that the BCAS mice showed a working memory deficit suggestive of the damage in frontal-subcortical circuits which is often seen in SIVD patients. ¹³ To date, BCAS mice have been relatively well recognized as one of the useful experimental models for VCI with white matter pathology, i.e., VCI associated with SSVD.^14,15 Moreover, novel mechanisms and potential therapeutic targets for white matter damage due to chronic cerebral perfusion have been studied using this mouse model. This review article exclusively focuses on this BCAS mouse model because of the increasing number of papers with the BCAS model. But of course, besides this BCAS model, there are multiple useful rodent models of vascular cognitive impairment. Table 1 summarizes the main advantage and disadvantage of the BCAS model over other rodent models of VCI, but the readers are encouraged to read the review articles that summarize and compare these models.^15–21 In addition, within in this review article, we briefly introduce other “stenosis-based” rodent VCI models and discuss what points are different from the mouse BCAS model.

Table 1.

Other rodent models of vascular cognitive impairment and dementia.

Animal	Model	Procedure/mechanism	Advantages of BCAS model	Disadvantages of BCAS model
Rat	SHRSP	Genetic model for hypertension	Genetic studies can be performed using knock-out or knock-in or transgenic mice	To study hypertension and stroke, SHRSP is more useful than BCAS model with WT mice.
Rat	BCCAO	Surgical occlusion of bilateral CCA	The visual pathway is not damaged.	Surgery is harder than BCCAO because of the small size of the vessels and organs.
Rat and Mouse	2-VGO	Surgical placement of ACs to CCA	Surgery is easier and device costs are cheaper.	The reduction rate of CBF is relatively rapid.
Mouse	ACAS	Surgical placement of ACs to Rt CCA and MCs to Lt CCA	Surgery is easier and device costs are cheaper.	The reduction rate of CBF is relatively rapid.
Rat and Mouse	Focal Injection of Vasoconstrictors	Direct injection of vasoconstrictor agents into the white matter	Global hypoperfusion including white matter can be made suitable for SIVD research.	To study white matter strokes such as lacunar infarcts, BCAS is inferior.
Mouse	Transgenic lines for SVD	Genetically inherited CADASIL or CAA	Suitable for sporadic SIVD studies. BCAS can be applied to these transgenic mouse lines.	Not suitable for studies of SIVD caused by inherited SVD. Surgical intervention is needed.
Rat and Mouse	MCAO	MCA occlusion	To study SIVD, BCAS model is more useful.	To study PSD, MCAO is more useful than BCAS model.

BCAS: bilateral common carotid artery stenosis; SHRSP: stroke-prone spontaneously hypertensive rats; WT: wild type; BCCAO: bilateral common carotid artery occlusion; CCA: common carotid artery; VGO: vessel gradual occlusion; AC: ameroid constrictor; CBF: cerebral blood flow; ACAS: asymmetric common carotid artery surgery; Rt: right; Lt: left; SIVD: subcortical ischemic vascular dementia; SVD: small vessel disease; MCAO: middle cerebral artery occlusion; MCA: middle cerebral artery; PSD; poststroke dementia.

Search and selection of literature

The relevant studies from 2004 to 2021 were searched (by HI and KA, individually) using the term ‘bilateral carotid artery stenosis or BCAS’, ‘cerebral hypoperfusion’, and ‘mouse or mice’ on PubMed. With this initial search, we found 119 papers. Among these papers, we manually excluded 23 papers because they did not use BCAS mice. After that, we carefully checked the rest of the 96 papers, and then 19 papers were newly added to our list because these papers were cited in the 96 papers. In the end, a total of 115 papers were reviewed for this review article (Figure 1). This search strategy and study exclusion criteria were pre-registered. As shown in Figure 2(a), the number of papers with the BCAS mouse model has been gradually increasing since the model was first reported in 2004 from Japan ¹² (Figure 2(a)). Especially, there has been a high increase in the recent five years (Figure 2(a)), and this may be partly because of the expansion of reporting countries (Figure 2(b) and (c)).

Figure 1.

Flowchart of literature selection. We used terms for the initial search as follows; ‘bilateral carotid artery stenosis’ or ‘BCAS’, ‘cerebral hypoperfusion’ and ‘mouse’ or ‘mice’ on Pubmed. One hundred nineteen papers were selected by the initial search. Twenty-three papers were excluded because BCAS mice were not used in them. After assessing each paper, 19 papers with data using BCAS mice were added to the reference list. The final 115 papers were used in this study.

Figure 2.

Publications using bilateral carotid artery stenosis mouse model. The number of publications using the bilateral carotid artery stenosis mouse model has been increasing (a). From 2004 to 2016, this model was used mainly in Japan and the US (b). Recently, this mouse model was used in various countries (c).

Methods of BCAS operation

The original BCAS papers specified the mouse strain and sex/age, duration of hypoperfusion, operation procedure, and micro-coil diameter for the BCAS model in detail.^12,13,22 Male C57BL/6J mice (age of 2 to 3-month-old, body weight, 24–29 g) are considered the most established mice for this model since other strains may have greater variability of cerebral blood flow (CBF) after cerebral hypoperfusion by the BCAS operation. The procedure of BCAS surgery is as follows: First, an anesthetized mouse with gas anesthetics should be placed in a dorsal recumbent position on the operating board with a heating pad. Then shave the ventral neck area, sanitize with 70% ethanol, and make a 1.0–1.5 cm midline skin incision in the neck. Remove the underlying fat and move the salivary glands with forceps to maximize the operating field. Expose both common carotid arteries from their sheaths under an operating microscope. Separating the carotid from the vagus nerve is important, and attention should be paid not to damage the nerve. Sutures should be located around the common carotid artery (CCA) for lifting the vessels and twine the micro-coil by rotating it around both CCA (Figure 3). Close the incision with fine sutures and return the mouse to the animal holding area. After awake, the BCAS mouse should move almost normally. In the reports from the group that established this mouse model, CBF decreased to about 68% from the baseline after 10 minutes and 84% after one month after BCAS operation when 0.18 mm inner diameter micro-coils were used. ²²

Figure 3.

Operation of bilateral carotid artery stenosis. After the skin is cut, blunt dissection with a small clamp or forceps is conducted until the trachea can be seen. Expose both common carotid arteries from their sheaths under an operating microscope. Separating the carotid from the vagus nerve (green arrows) is very important and attention should be paid not to damage the nerve. Sutures should be located around the CCA for lifting the vessels (a). Then twine the microcoil (yellow) by rotating it around the left CCA (b) and right CCA (c).

To date, several modifications have been made. The modifications of the BCAS methodology are summarized in Table 2.^{12,13,23–53} The major modified points include mouse strain, mouse age at surgery, and micro-coil diameter (Supplementary Figure S1). Originally, the BCAS model was proposed to use the C57BL/6J strain, as CBF in other strains may have variability due to the difference in vascular structures. ²² In recent studies, however, at least B6D3F1 and FVB strains are confirmed to show similar CBF reduction after BCAS surgery to C57BL/6J mice.^39,42 As for mouse age, the original BCAS paper by Shibata et al. used 2-month-old mice, and most studies used 2 to 4-month-old mice. However, some studies used older mice (Table 2). The oldest mouse age for BCAS studies is 22-month-old, and even in the aged mice, approximately 15% CBF reduction was observed 30 days after the BCAS operation. ⁴⁶ But it should be noted that the baseline CBF of 22-month-old mice was already decreased to about 60% compared to 3-month-old mice. Therefore, when we use older mice for BCAS studies, we may need to pay attention to the baseline CBF of those mice. As for the micro-coil issue, while a majority of BCAS studies used the 0.18 mm inner diameter micro-coils for both CCA, some studies used micro-coils with different diameters for each carotid artery.^{23,32,35,38,41} To date, 0.18 mm coils are considered most appropriate to study white matter lesions caused by chronic hypoperfusion, but other sizes of micro-coils (e.g., 0.16 ∼ 0.22 inner diameter) were often used. However, the 0.16 mm micro-coils would cause significant gray matter changes and a high mortality rate (75% in 14 days after surgery) compared to 0.18 mm micro-coils (the average mortality rate with 0.18 mm coils is 12.4% (0%–29.2%).^{12,36,48,54–67} On the other hand, the larger sizes of micro-coils (>0.20 mm inner diameter) may not properly induce prolonged cerebral hypoperfusion. ¹² Therefore, when we use other sizes of micro-coils than 0.18 mm micro-coils, we should carefully check CBF after BCAS operation. In addition, we may also need to consider the influences of manufacturer differences for micro-coils. Shibata et al. obtained micro-coils from Sawane Spring for their original BCAS study, but there are several manufacturers for micro-coils these days (Table 2). Recent data indicate a relatively low mortality rate even in the 0.16 mm coils that range from 25% to 37.5%, and this discrepancy may be partly because of the manufacturer differences. The modified method of BCAS is expanding, and researchers now choose age at operation, duration of hypoperfusion, and coil diameter according to the purpose of their studies.

Table 2.

The modifications of BCAS methodology.

Year, first author	Background strain	Age (Month)	Sex	Hypoperfusion duration (days)	Micro-coil diameter (mm)	Coil manufacturare (country)	Mortality rate (coil)	Post-BCAS CBF(coil, days)	Method of CBF measurement
2004, Shibata (Original model)	C57BL/6	2.5–3	Male	30	0.16, 0.18, 0.20, 0.22	Sawane spring (Japan)	75% (0.16), 15% (0.18)	81% (0.16, 30) 88% (0.18, 30)	Laser-Doppler flowmetry(LDF)
2007, Shibata (Original model)	C57BL/6	2.5–3	Male	30	0.18	Sawane spring (Japan)	NA	NA	NA
2009, Miki	C57BL/6	4–4.5	Male	35	left 0.16, right 0.18	Sawane spring (Japan)	18.80%	60–70% 27	LDF
2010, Nishio	C57BL/6	4	Male	240	0.18	Sawane spring (Japan)	NA	85% 90	Laser speckle imaging (LSI)
2011, Coltman	C57BL/6	3–4	Male	28	0.18	Sawane spring (Japan)	NA	NA	–
2011, Reuner	C57BL/6	3–4	Male	3, 28	0.18	Sawane spring (Japan)	NA	NA	–
2011, Holland	C57BL/6	3–4	Male	28	0.18	Sawane spring (Japan)	NA	NA	–
2012, Okamoto	C57BL/6	2.5, 4, 5	Male	56, 84	0.18	Sawane spring (Japan)	NA	85% 84	LSI
2013, Miyamoto	C57BL/6	2, 8	Male	3, 7, 14	0.18	Sawane spring (Japan)	NA	NA	–
2014, Hattori	C57BL/6	5–9	Male	28	0.18	Sawane spring (Japan)	NA	83% 28	LSI
2014, Toyama	C57BL/6	2	Male	28	0.16, 0.18	Sawane spring (Japan)	37.5% (0.16), 0% (0.18)	73% (0.16, 28) 80% (0.18, 28)	LSI
2014, Yata	C57BL/6	2–4	Male	1, 14	right 0.16, left 0.18	Sawane spring (Japan)	NA	40–50% 14	Two-photon microscopy (pial artery)
2016, Saggu	C57BL/6	2.5–4	Male	7, 42	0.17	Sawane spring (Japan)	NA	75% 7	LSI
2017, Kitamura	C57BL/6	4–5	Male	84	0.18	Sawane spring (Japan)	NA	50–60% 84	MRI (ASL, corpus callosum)
2017, Tsai	C57BL/6	3	Male	30	left 0.16, right 0.18	Sawane spring (Japan)	NA	70% 30	Laser Doppler imaging (LDI)
2017, Wolf	C57BL/6	3, 19	Female	49	0.18	Wuxi Samini spring (China)a	20%	NA	–
2017, Boehm-Sturm	C57BL/6	9–13	Male	49	0.16	Shannon Coiled Spring (Ireland)	NA	75% 28	MRI (ASL)
2018, Fowler	C57BL/6	3–4	Male	7	right 0.16, left 0.18	Sawane spring (Japan)	19.40%	45% 7	LSI
2018, Song	FVB/N	2–2.5	Male	42	0.18	Sawane spring (Japan)	NA	60–70% 42	Two-photon microscopy (capillaries)
2018, Toyama	C57BL/6	2	Male	28	0.16	NA	NA	70–75% 28	LSI
2018, Roberts	C57BL/6	3	Male	7, 14, 30	0.18	Waken B Technology (Japan)	NA	NA	–
2019, Gao	C57BL/6	2–2.5	Male	28	0.18, 0.20	Sawane spring (Japan)	12%(0.18)	NA	–
2019, Shimada	B6D2F1	8	Male	28	0.18	Sawane spring (Japan)	NA	NA	–
2020, Foddis	C57BL/6	2–3	Male	2, 8	0.16	Shannon Coiled Spring (Ireland)	NA	72% 1	MRI (ASL)
2020, Sigfridsson	C57BL/6	6	Male	42	0.18	Sawane spring (Japan)	NA	NA	–
2021, An	C57BL/6	6–8	Male	30	0.16	RuikeBiotech (China)	25%	65% 15	LDF
2021, Baik	C57BL/6	3, 22	Male	7, 30	0.18	Sawane spring (Japan)	NA	80–85% 30	LSI
2021, Liu	C57BL/6	2–3	Male	30	0.18	Wuxi Samini spring (China)a	NA	75% 30	LSI
2021, Liu	C57BL/6	4	Male	28	0.18	Sawane spring (Japan)	23.40%	65% (0)	Laser Doppler perfusion monitoring (LDPI)
2021, Messerschmidt	C57BL/6	2–3	Male	7, 28, 49	0.16	Shannon Coiled Spring (Ireland)	NA	NA	–
2021, Poh	C57BL/6	3–4	Male	1, 3, 7, 15, 21, 30 ,60	0.18	Sawane spring (Japan)	NA	80% 30 , 95% 60	LSI
2021, Ohtomo	C57BL/6	8	Male	42	0.18	Samini (Japan)a	NA	NA	–

The modified points were red-colored. MRI: magnetic resonance imaging; ASL: arterial spin labeling, NA: not available.

^aWuxi Samini and Samini are affiliates of Sawane spring.The original model was described in bold.

Cerebral blood flow

Cerebral blood flow (CBF) after BCAS have been measured by multiple techniques, and in this section, we introduce CBF changes after BCAS operation (Table 3).^{12,13,23,24,28–31,33,34,38,48,50,55–57,61,62,64,66,68–89} Laser doppler flowmetry (LDF) and laser speckle imaging (LSI) are the most widely used method for assessing CBF in BCAS mice. The relative CBF of the cortex can be measured by LDF. ⁹⁰ The LSI is a technique for recording CBF that can be used to acquire semiquantitative maps of blood flow in vasculature on the cortical surface in animal models. ⁹¹ The advantage of LSI is the relative simplicity and cost-effectiveness of its instrumentation for standalone systems. ⁹² The CBF after BCAS showed similar change between LDF and LSI mainly because those techniques are both assessing the blood flow of the cortical surface. According to the CBF data measured by LDF or LSI, CBF decreases to about 60% from baseline after the micro-coil placement and then gradually recovers to around 70% and 80% seven days and 25 ∼ 30 days after BCAS operation, respectively. ASL measures blood flow directly and suffers from a low signal to noise ratio, but blood oxygen level dependent (BOLD) is based on functional MRI (fMRI) where it is used to localize the hemodynamic response to neural activity. ⁹³ While some fMRI studies BOLD have been conducted in human brain with small vessel diseases,^93,94 BOLD-fMRI studies using BCAS model mice have not been reported yet. This is partly due to the technical difficulty in taking BOLD-fMRI images using mouse brains. However, some studies have reported its usefulness, ⁹⁵ and imaging data of neurovascular coupling or cerebrovascular reactivity to carbon dioxide in BCAS mice would help to assess the influence of collaterals to small vessel blood flow and to estimate the comparability of the human data.

Table 3.

Cerebral blood flow change after bilateral carotid artery stenosis operation.

	Day 0	Day 1–3	Day 7	Day 14	Day 25–30	Day 50–60	Day 80–90
Laser Doppler Flowmetry	61%48–77	73%72–75	73%65–77	70%65–79	79%60–89	76%70–85	85%
Laser Speckle Imaging	61%50–69	60%40–75	71%45–82	83%80–88	80%70–90	85%	NA
MRI (Atrial Spin Labeling)	NA	50%	53%50–55	50%	70%	NA	50–60% (thalamus, corpus callosum)

Average (range) cerebral blood flow at each timepoint was shown in this table.

On the other hand, CBF measured by atrial spin labeling (ASL) on MRI showed a little different tendency. After BCAS, the CBF dropped to around 50% by day 14, then recovered to 70% on days 25 ∼ 30. One study focusing on CBF in the thalamus and corpus callosum showed CBF reduction to 50–60% from the baseline three months after BCAS. ³⁴ The main difference compared to LDF/LSI is that CBF in the whole brain or deep area can be measured on MRI. A study using ASL indicated that cortical and subcortical parenchymal CBF levels are continuously decreased for at least 14 days. ⁷³

In vivo imaging using two-photon laser-scanning microscopy in BCAS was also performed to assess deep cortical capillary flow. ³² Inflow velocity of the pial arteries and outflow velocity of the pial veins decreased about 50% from the baseline after BCAS from day 1 to day 14 by repetitive line-scanning. Another study with in vivo two-photon microscopy observation of penetrating arteries has shown that the velocity of blood flow of BCAS mice was 62.5% of sham-operated mice six weeks after the operation. ³⁹ A recent study with in vivo two-photon microscopy reported comparative data of CBF between cortex and corpus callosum. In BCAS mice, red blood cell (RBC) flux in the white matter (e.g., corpus callosum) was reduced significantly in comparison to the controls, while RBC flux in the gray matter (e.g., cortex) was preserved. The results suggested that blood flow in the corpus callosum may be less efficiently regulated when challenged by physiological perturbations as compared to the cortex. ⁹⁶ The CBF assessed by LDF or LSI returns to around 80% from the baseline one month after BCAS. In contrast, CBF in deeper tissue or capillary may be lower than the cortical CBF evaluated by LDF or LSI. Micro-heterogeneity of flow in BCAS mice was revealed by optical coherence tomography. ⁹⁷

In summary, the reduction rate of CBF in brain surface is 30–40% at 3 days and recover to 20% at 1 month probably due to collateral circulation. The CBF reduction in the deeper area may be more severe, estimated reduction rate is 30% 1 month after surgery. Future studies are warranted to carefully examine the local supply heterogeneity at the capillary level, even at non-ischemic global flow levels.

Cognitive function

Thus far, several behavioral tests have been used to assess the cognitive function of BCAS mice (Table 4). 13^,14^,23^–25^,29^,34^,37^,52^,54^,56^,57^,61^,70^,72^,75^,76^,78^,79^,81^,83^, 98^–111 For example, in the 8-arm radial maze test, which was used in the original study, spatial working memory declined in BCAS mice (2.5-month to 5-month-old) with 28 to 6 months of hypoperfusion duration. Reference memory was intact in 2.5 to 3-month-old mice with 1-month BCAS duration. In the Y-maze test, BCAS mice (2-month to 4-month-old) with hypoperfusion duration of 28–60 days tend to show spatial working memory decline. Furthermore, another study using 4-month-old mice with 5-month hypoperfusion (behavioral test at 9-month-old) also showed working memory impairment compared to sham mice in the 8-arm-radial maze test. ²⁴ A shorter duration (e.g., 14 days) of hypoperfusion may not show a cognitive decline compared to the control group.^29,76 Another study confirmed that 14-day hypoperfusion did not cause spatial working memory deficits (assessed by the Y-maze tests) in 2-month-old mice, ²⁹ but this study also showed that in 8-month-old mice, 14-day hypoperfusion did induce cognitive decline, ²⁹ indicating that aging may be an important risk factor for cerebral hypoperfusion-related cognitive dysfunction in mice. However, the data regarding cognitive function in aged BCAS mice is still insufficient in both Y-maze and 8-arm-radial maze tests.

Table 4.

Behavioral test results in mice with prolonged hypoperfusion induced by bilateral carotid artery stenosis.

Behavioral test	Mouse age at BCAS (month)	Hypoperfusion duration	Results	Studies (reference number)
Y-maze	2–3	30 days	Spatial working memory ↓	Hou, 2015 (98)
	2–2.5	60 days	Spatial working memory ↓	Han, 2019 (61)
	2.5–3	14 and 28 days	Working memory → at day 14, ↓ at day 28	Miyanohara, 2018 (76)
	2.5	30 days	Spatial working memory↓	Washida, 2010 (70)
	2.5	28 days	Spatial working memory ↓	Miyamoto, 2013 (72)Miyamoto, 2020 (99)
	2.5	4 and 6 months	Working memory ↓ (both timepoints)	Khan, 2018 (78)
	3	14 days	Spatial working memory ↓	Dong, 2011 (100)
	3	21 days	Spatial working memory ↓	Dong, 2011 (100)
	3–4	28 days	Spatial working memory ↓	Koizumi, 2018 (79)Du, 2020 (83)
	2 and 8	14 days	Working memory of middle-aged BCAS mice↓ (compared to young BCAS mice)	Miyamoto, 2013 (29)
8-arm radial maze	2.5–3	28 days	Spatial working memory ↓Reference memory →	Chen, 2017 (56)Chen, 2019 (101)
	2.5–3	30 days	Spatial working memory↓Reference memory →	Shibata, 2007 (13)
	2.5	30 days	Spatial working memory ↓	Duan, 2009 (54)
	< 3	6 months	Spatial working memory ↓	Holland, 2015 (14)
	3–4	2 months	Spatial working memory ↓	Coltman, 2011 (25)
	4	5 months	Spatial working memory ↓	Nishio, 2010 (24)
	4–5	3 months	Spatial working memory ↓	Kitamura, 2017 (34)
Barnes maze	4	6 months	Reference memory ↓	Nishio, 2010 (24)
Water maze	2	28 days	cognitive function ↓	Park, 2019 (102)
	2–2.5	14 days	Spatial learning memory ↓	Yuan, 2017 (103)
	2.5	42 days	Spatial learning memory ↓	Iwanami, 2015 (104)
	2.5	42 days	Spatial learning memory ↓	Mogi, 2018 (105)
	3	10-14days 24-28days	Spatial learning memory ↓ exception on day 26–28	Ahn, 2016 (106)
	< 3	6 months	Spatial reference lerning and memory ↓	Holland, 2015 (14)
	3–4	1 month	Reference memory →	Coltman, 2011 (25)
	4–4.5	35 days	Reference memory ↓	Miki, 2009 (23)
	8	7 weeks	Reference memory →	Ohtomo, 2021 (52)
	9–13	7 weeks	Spatial learning memory ↓	Boehm-Strum, 2017 (37)
NORT	2	28 days	Cognitive function (30 min retention) ↓	Takase, 2021 (107)
	2–2.5	28 days	Cognitive function (6 h retention) ↓	Temma, 2017 (108)
	2–2.5	60 days	Cognitive function (1 h retention) ↓	Han, 2019 (61)
	2–3	28 days	Recognition memory (6 h retention)↓	Khan, 2015 (109)Kakae, 2019 (110)
	2.5–3	28 days	Working memory (6 h retention) ↓	Miyanohara, 2018 (76)
	2.5–3	28 days	Cognitive function (1 h retention) →	Lee, 2019 (81)
	2.5–3	40 days	Short-term (90 min) memory ↓Long-term (24 h) memory →	Patel, 2017 (57)
	3	5 weeks	Cognitive function (1 day retention) ↓	Dominguez, 2018 (75)
	3	90 days	Working memory (15 min retention) ↓	Tsai, 2015 (111)
	3–4	28 days	Cognitive function (30 min retention) ↓	Koizumi, 2018 (79)
	8	7 weeks	Cognitive function (30 min retention) ↓	Ohtomo, 2021 (52)
	9–13	7 weeks	Short-term (2 h) memory →	Boehm-Strum, 2017 (37)

In the Morris water maze (MWM) test, 2 ∼ 3-month-old mice with 14-day ∼ 6-month cerebral hypoperfusion tend to show cognitive dysfunction. One study using 3 ∼ 4-month-old mice with a BCAS duration of one month showed no significant cognitive decline compared to the control group. ²⁵ Another study reported compensatory mechanisms of cognitive recovery on days 26 ∼ 28 post-BCAS surgery. ¹⁰⁶ Their MWM test data showed a decline of spatial working memory after 10 ∼ 14 days or 24 ∼ 25 days after BCAS, but the cognitive function did not show a significant difference compared to the sham mice on days 26 ∼ 28 after BCAS. Middle-aged mice (8 ∼ 13-month-old) showed various results regarding cognitive function.^37,52 In the novel object recognition test (NORT), most studies indicated that BCAS mice (2 ∼ 8-month-old) with hypoperfusion duration of 28 ∼ 90 days show a short-term (15 minutes to 6 hours) memory decline. On the other hand, some studies did not replicate this tendency.^37,81 One study analyzed the discrimination rate of the NORT in 3-month-old and 21-month-old female mice and reported that there were no significant cognitive differences in the discrimination rate between the two age groups. ³⁶ The protocol with a long-retention interval (e.g., >24 hours) or aged mice may not detect cognitive decline by cerebral hypoperfusion.^37,57

Overall, young mice seem easier to be detected cognitive decline by prolonged cerebral hypoperfusion. This is partly because aged mice exhibit some cognitive impairment at baseline, ¹¹² and the deleterious effects of cerebral hypoperfusion on cognitive function may sometimes be difficult. However, how aging affects cognitive decline by cerebral hypoperfusion is still to be elucidated.

Pathophysiology in corpus callosum, cortex, and hippocampus

Histopathological examination detects changes in protein expression caused by cerebral hypoperfusion in BCAS mice. The authors, who developed this BCAS model, reported that the most vulnerable brain region by cerebral hypoperfusion was white matter (e.g., corpus callosum), in which the proliferation of activated microglia and astroglia was observed three days after BCAS. ¹² The smaller changes were also observed in the caudoputamen, internal capsule, and anterior commissure. The white matter lesion is estimated from the reduced intensity of myelinated fibers and the compromised integrity of the myelin with relatively intense change in the medial part of the corpus callosum on Klüver-Barrera (KB) staining 14 days after BCAS. Although the gray matter is almost intact one month after BCAS when 0.18 mm micro-coils are used, ischemic lesions would extend from white matter to the hippocampus and cortex when 0.16 mm diameter micro-coils were used.^12,23 A duration of 8 months with cerebral hypoperfusion causes the development of pyknotic neurons in the cortex and hippocampus. ²⁴ Hippocampal atrophy is also observed in long-term cerebral hypoperfusion in mice. ¹¹³ The main cause of hippocampal atrophy is thought to be secondary to the proceeding damage of white matter.

A recent study has shown that there are no significant changes in MAP2 expression and NeuN positive neuron numbers in the cortex and hippocampus (CA3) at three days, ten days, one month, and three months after BCAS compared to the sham group. ¹¹⁴ On the other hand, some studies reported histological changes in the hippocampus and cortex after BCAS. Mice with cerebral hypoperfusion showed impairment of synaptic plasticity confirmed by assessing reduced spine density in both hippocampus and cortex one month after the BCAS surgery. ¹¹⁵ In addition, one study using ex-vivo MRI revealed a slight injury to the hippocampus on T2 weighted images (WI) and significantly lower fractional anisotropy (FA) value on diffusion tensor imaging (DTI) metrics measurement. ⁴⁷ Astrogliosis (increased GFAP positive cells) and microglial activation (increased Iba1 positive cells) were also reported in the hippocampus in mice 30 days after BCAS without neuronal loss in the hippocampus and cortex. In addition, changes in the blood-brain-barrier (BBB) and BBB-associated extracellular matrix would occur shortly after BCAS in the hippocampus, cortex, and striatum. ⁵³ These studies indicate that the inflammation or microstructural changes would appear in brain regions besides the corpus callosum shortly after BCAS, which might underlie the gradual development of BCAS non-white matter pathology.

The selective white matter damage by 4-week cerebral hypoperfusion in BCAS mice using 0.18 mm micro-coils resembles the pathology of human SIVD. In contrast, BCAS mice with cortical or hippocampal changes induced by long-term hypoperfusion (e.g., longer than 6-month cerebral hypoperfusion) or by narrower inner diameter micro-coils could be considered a mouse model for the broader spectrum of VCI related to SVD and carotid artery stenosis in human. Therefore, depending on the purpose, we could manipulate the brain region and the degree of hypoperfusion-induced damage by adjusting the conditions of BCAS. However, as noted above, brain damage by cerebral hypoperfusion would be affected by aging and mouse strains, so each laboratory needs to carefully optimize the model before starting its projects.

Mechanisms of brain damage by cerebral hypoperfusion

Oxidative stress, microvascular injury, excitotoxicity, BBB dysfunction, and secondary inflammation are considered the main mechanisms of tissue damage by prolonged cerebral hypoperfusion. Hypoxic conditions due to cerebral hypoperfusion would activate several deleterious cascades. For example, hypoperfusion induced by BCAS causes BBB disruption with vasogenic edema, damaging myelinated fibers of the deep white matter and extravasation of serum proteins. ¹⁶ Moreover, stagnation of capillary flow in the cortex induced by a plug with leukocyte has been observed on in vivo two-photon laser-scanning microscopy. ³² In addition, in vitro studies combined with in vivo studies with BCAS mice have revealed the role of glial cells and cell-cell interactions under hypoxic conditions.^{29,31,40,42,58,62,64,72,74,83,85,99,116–122} In this section, we mainly focus on the mechanisms of how oxidative stress, vascular injury, and inflammatory responses cause glial pathology in BCAS mice.

Reactive oxygen species (ROS) levels increase in damaged white matter, along with the suppression of OPC-to-oligodendrocyte differentiation and loss of myelin staining. ⁷² Oxidative stress-apoptosis signal-regulating kinase 1 (ASK1)-p38 cascade also plays a pathogenic role through BBB breakdown via the disruption of endothelial tight junction proteins.^31,122 The activation of matrix metalloproteinases (MMPs) by hypoxia has been demonstrated to be involved in BBB opening via disrupting basal lamina and tight junction proteins,^116,123 and MMPs are thought to play a critical role in the BBB disruption, glial cell activation, and white matter lesions in BCAS mice.^{61,68,116,124} In fact, the expressions of MMP-9 and MMP-2 increase were confirmed in mice after the BCAS operation.^61,68,116 Inflammatory changes would also play an important role in the pathogenesis of cerebral hypoperfusion. The number of reactive astrocytes and activated microglia increases in the corpus callosum 14 days after BCAS, and these phenomena continue up to at least 30 days. ⁵⁴ Proinflammatory cytokines, including tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6, also increase in the corpus callosum after BCAS.^76,103 Activated microglia in brain is the major source of cytokine release under pathological conditions, and these inflammatory profiles are exacerbated by adenosine A_2A receptor (A_2AR) deficiency.^54,125 The protective effects of A_2AR inactivation on the acute ischemic brain are thought to be caused by the reduction of glutamate outflow and excitotoxicity-related damage,^126,127 while A2AR inactivation may be associated with increased expression of proinflammatory cytokines in chronic cerebral hypoperfusion induced by BCAS. ⁵⁴ In addition, cerebral hypoperfusion induced by BCAS activates the absence in melanoma 2 (AIM2) and NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome. ¹²⁸ The inflammasome is an intracellular multiprotein complex that initiates an innate immune response in neurodegenerative diseases. ¹²⁹ Recent studies indicated that the AIM2 inflammasome mediates hallmark neuropathological alterations in BCAS mice.^50,51 These studies suggest that cerebral hypoperfusion initiates complex molecular and cellular inflammatory pathways that contribute to long-term cognitive impairment.

To find a novel mechanism for brain damage after cerebral hypoperfusion, a few recent studies have provided DNA array or RNA-sequencing (RNA-seq) data for clarifying transcriptome profiles related to cerebral hypoperfusion.^{26,46–48,77,107} A microarray analysis using white matter samples indicated alterations in biological pathways, including inflammatory responses, cytokine-cytokine receptor interactions, blood vessel development, and cell proliferation processes. ²⁶ A recent RNA-seq study using corpus callosum samples from BCAS mice showed significant activation of oligodendrogenesis pathways along with angiogenic responses at four weeks after cerebral hypoperfusion, ¹⁰⁷ indicating that cerebral hypoperfusion may not always cause brain damage, but under some conditions, could activate compensative responses. Another recent study examined the transcriptome profile in the hippocampus in BCAS mice. BCAS-induced changes in hippocampal gene expression differed between young (3-month-old) and aged (22-month-old) mice. ⁴⁶ The transcriptomic analysis of this study indicated that in comparison to young sham mice, many pathways altered by BCAS in young mice resembled those already present in sham-aged mice. In addition, over 30 days after the onset of cerebral hypoperfusion, aged BCAS mice showed minimal effect on hippocampal gene expression. Another study that analyzed astrocytic transcriptional profiles with bulk RNA-seq revealed a significant upregulation of classic transcriptomic signatures of reactive astrocytes, including pan-reactive signatures (Vim, Lcn2, Cd44, Steap4, Cxcl10, Gfap, Serpina3n, Osmr, Timp1), clustered A1 phenotype genes (Fkbp5, Gbp2, H2-D1, H2-T23, Iigp1, C3, C4b, Psmb8, Serping1, Srgn) and A2 phenotype genes (Cd14, Emp1, Ptgs2, Tgm1). ⁴⁷ In recent years, many studies have focused on transcriptomics regarding the brain vasculature,^130,131 but the transcriptome data are still not satisfactory in the BCAS model, and detailed information regarding gene and protein expression profiles for endothelium and perivascular cells using BCAS mice is awaiting for better understanding of which function is regulated at different levels of the vascular tree and how homeostasis is affected by cerebral hypoperfusion.

Comparison with other “stenosis-based” rodent models of cerebral hypoperfusion

After the development of the mouse model of cerebral hypoperfusion by BCAS, several rodent hypoperfusion models were reported. For example, rats with hypertension subjected to bilateral common carotid artery stenosis using ameroid constrictors called spontaneous hypertensive rat two-vessel gradual occlusion (SHR-2VGO) was reported in 2016. ¹³² The SHR-2VGO shows slowly evolving white matter abnormalities and subsequent spatial working memory impairment, which replicates some selective aspects of the pathophysiology of SIVD in humans. Although SHR-2VGO may be a reasonable rat model for studying SIVD, the lack of knock-out or transgenic rats to conduct genetic studies makes it challenging to explore potential therapeutic targets in disease animal models with cerebral hypoperfusion. In mice, a gradual common carotid artery (GCAS) mouse model shows a slower reduction of CBF than BCAS model which resembles a condition in the human brain with chronic cerebral hypoperfusion due to aging and cardiovascular risk factors. ¹³³ In the GCAS model, ameroid constrictor devices which can cause gradual narrowing the diameter are applied to bilateral carotid artery instead of microcoils. In addition, an asymmetric common carotid artery stenosis (ACAS), in which an ameroid constrictor is used on one side of the common carotid artery and a microcoil is used on the other side, was also reported recently. ¹³⁴ The mortality rate of ACAS was 20%, ¹³⁴ which is slightly higher than the BCAS model. One of the drawbacks of the BCAS model is a relatively acute reduction of CBF after the surgery, but in both GCAS and ACAS models, the acute CBF drop would not be observed. According to the recent review article that provided the comparison between the BCAS and ACAS models, ¹⁷ the difficulty of surgery for ACAS is higher than BCAS, and the cost for BCAS is approx. $50 (microcoil $25 × 2) while the one for ACAS is $125 (ameroid constrictor $100 + microcoil $25). ¹⁷

Identifying potential therapeutic targets

The BCAS model has also been applied to disease mice, including Alzheimer’s disease (AD) mice, Parkinson’s disease (PD) mice, and diabetic mice. Chronic cerebral hypoperfusion by BCAS increased Aβ fibrils and induced Aβ deposition in the intracellular compartment in amyloid precursor protein (APP)-Tg AD mice, ¹³⁵ suggesting that cerebral hypoperfusion may accelerate the pathological changes of AD. Another study showed that BCAS-operated APP-Tg mice exhibited an increased aggregation of Aβ and significantly impaired learning ability compared to the sham group. ¹³⁶ A recent study supports the idea that cerebral hypoperfusion worsens AD pathology by showing that Aβ oligomers with high molecular weight increased in the brain of BCAS-operated mice. ⁸⁸ In the tau-Tg mice (T44), compared to sham tg mice, cerebral hypoperfusion by BCAS promoted the expression of phosphorylated tau in tg mice (T44). ⁴² On the other hand, this study also showed that the expression of leptin-receptor was upregulated by cerebral hypoperfusion. Because leptin demonstrates potential as a therapeutic target for CNS diseases, such as ischemic stroke and AD, cerebral hypoperfusion may activate endogenous repairing processes even in AD mice. Besides AD mice, hypoperfusion induced by BCAS exacerbates cognitive impairment in a mouse model of PD, ¹³⁷ proposing that maintaining adequate cerebral perfusion should be important for preventing cognitive decline in PD. Type 2 diabetic mice also show more severe white matter injury by cerebral hypoperfusion. The histopathological findings indicated that decreased proliferation and survival of OPCs may play an important role in the progression of white matter lesions after ischemia in diabetics. ¹³⁸ Because CBF reductions are known to be one of the early symptoms of CNS diseases, such as AD, ¹³⁹ the BCAS model combined with tg mice would be useful to examine the pathological mechanisms of other CNS diseases than SIVD for identifying a new therapeutic target.

The BCAS model could also be useful for drug testing. The number of studies that used potential therapeutic agents in BCAS-operated mice is increasing. These agents include MMP inhibitor, ¹¹⁶ TrkB antagonist, ¹⁰⁶ NKCC1 inhibitor, ¹⁴⁰ CSF-1R inhibitor, ¹⁴¹ an inhibitor of immunoproteasome subunit LMP7, ¹⁴² and ginsenoside Rg1 ⁶⁷ Drugs approved for other diseases such as telmisartan, ⁷⁰ edaravone,^72,118 cilostazol,^34,77 melatonin, ³⁵ tamoxifen, ¹⁰¹ levetiracetam, ⁸⁰ fingolimod ¹²⁰ and digoxin ⁸⁶ were confirmed to be effective in mitigating white mater damage and/or cognitive decline in BCAS mice. These studies provide proof of concept that these drugs could be re-purposed for SIVD or vascular-related dementia. In addition, recently, adrenomedullin has been introduced as one of the novel therapeutic candidates for VCI ¹⁴³ and using transgenic mice of circulating adrenomedullin overexpression, it was reported that adrenomedullin overexpression would work positively in both vascular function and cognitive function after cerebral hypoperfusion by BCAS. ⁷¹ Adrenomedullin was found to upregulate vascular endothelial growth factor and basic fibroblast growth factor through the adrenomedullin receptor and the phosphatidylinositol 3-kinase pathway, ¹⁴⁴ so future studies are warranted to test the efficacy of adrenomedullin-related drugs in BCAS mice.

Considering the fact that polypharmacy among elderly patients has become a social issue around the world, it may be important to pursue a therapeutic approach with a non-pharmacological strategy. In this regard, environmental control, including diet and physical intervention, is proposed as an important target for therapeutic intervention for SIVD and other vascular-related dementia, and interestingly, some studies have pursued this research direction. Environmental enrichment, defined as living with extra toys in addition to the standard housing, e.g., running wheels, hanging chains, igloos, and a paper tunnel, reduced glial damage/activation in BCAS mice. ¹⁴⁵ A limited rather than full-time exposure to environmental enrichment regimen also attenuates corpus callosum atrophy, greater white matter disintegrity, and working memory deficits induced by BCAS. ¹⁴⁶ A recent study also confirmed that BCAS-operated mice on intermittent fasting intervention, where food was provided for only 6 hours during the active phase, showed higher hippocampal neuronal density and spatial working memory compared to the BCAS-operated mice fed ad libitum. ¹⁴⁷ In addition, exercise with treadmill training was shown to ameliorate cognitive decline and increase the population of OPCs in the subventricular zone in young BCAS-operated mice. ⁸⁴ However, the same group also reported that the same exercise regimen showed milder effects on cognitive function in middle-aged BCAS- operated mice, ⁵² again supporting the idea that aging may be a critical factor in affecting the cerebral hypoperfusion-induced cognitive decline in BCAS mice.

Prospective for future BCAS studies

The BCAS model could be a powerful tool to clarify the pathological mechanisms in cerebral small vessel diseases. ¹⁵ However, there are some important points that we need to keep in mind for future BCAS studies. First, there is no perfect mouse model for SIVD, and indeed, the BCAS model has some limitations. Human SVID is mainly caused by small vessel disease (associated with fibrinoid necrosis, lipohyalinosis, fibrohyalinosis, microatheroma, and microaneurysm) resulting in obstruction of blood flow to the small vessels of the brain.^10,148,149 Whereas the BCAS model inducing hypoperfusion by blocking major extracranial vessels, such as the CCA. Although the brain pathologies of BCAS mice show the myelin loss and inflammatory changes in the white matter resembling human SIVD, arteriosclerosis is lacking in BCAS-hypoperfusion mice mainly because hypoperfusion is just induced by blocking major extracranial vessels (e.g. common carotid arteries). This is the major difference between the cause of hypoperfusion in human SIVD vs the BCAS model. But, because hereditary small vessel disease mouse models show similar mechanism of human small vessel disease ¹⁵⁰ and the major risk factors of SIVD in humans are aging and hypertension, using these mouse models for BCAS experiments may partly overcome this limitation of the BCAS model. Another potential difference between human SIVD and the BCAS model is a relatively sharp CBF reduction in the early phase after BCAS, which makes this model hard to completely mimic the chronic hypoperfusion conditions observed in human SIVD. Although low CBF is associated with white matter hyperintensity on MRI in the human brain, the decline of CBF happens gradually without apparent symptoms in elderly people with cerebral small vessel disease. ¹⁵¹ In addition, MR angiography data in BCAS mice revealed increased tortuosity in the Circle of Willis at 7 weeks after the operation. ³⁷ The increased tortuosity may be partially responsible for the recovery in CBF because this collateral structure attempts to compensate. Another study has revealed the promptness of external carotid artery retrograde flow recruitment together with posterior communicating artery patency influence ischemic lesion volume in BCAS mice. ⁴³ Although how the collaterals influence CBF of small vessels is still unknown, there may be some different mechanism in blood flow recovery after cerebral hypoperfusion between humans SIVD and the BCAS model. Therefore, as a few studies have been conducted, a comparison with human samples would provide important information to interpret the findings in this model.^{28,119,128,152}

Second, we have to pay attention to interpret the data of trial therapies in the BCAS mice model. The original BCAS paper used young male mice, and we still do not have sufficient data regarding how sex and age affect the mechanisms of brain damage and repair after cerebral hypoperfusion. Because there are significant differences in cellular function in the human tissue, including the brain, between males and females ¹⁵³ and because aging is an important risk factor for most CNS diseases, including vascular-related diseases, ¹⁵⁴ the influence of sex and age should be carefully considered to assess the effect of pharmacological or environmental intervention in white matter damage and cognitive dysfunction by cerebral hypoperfusion for future studies. Furthermore, as discussed in the previous paragraph, the blood flow reduction of the majority of SIVD due to sporadic small vessel diseases in human is caused by in-situ arteriosclerosis, while the blood flow reduction of small vessels in BCAS model is secondary to carotid artery stenosis. Unlike real SIVD patients, the influence of vascular risk factors including hypertension, hyperlipidemia, and diabetes are not considered in BCAS model unless we perform BCAS operation to mice with vascular risks. This should be kept in mind when we interpret the data of trial therapies in a BCAS hypoperfusion model. As cerebral amyloid angiopathy model mice with cerebral hypoperfusion by BCAS operation showed cortical microinfarcts and cerebral microbleeds, which are known as major pathological changes of cerebral small vessel disease,^28,155,156 certain transgenic mice or mice with comorbidities will be useful for future studies for BCAS studies to obtain translational data of biological and imaging markers from animals to human beings.

And finally, in terms of identifying new therapeutic targets for SIVD, transcriptome data may be a key point for future BCAS studies. The transcriptome profile of BCAS-operated mice would provide a valuable dataset for assessing new therapeutic targets and searching for new biomarkers in CNS disorders. For example, changes in the extracellular matrix are recently attracting attention in small vessel diseases.^157–160 The term matrisome has been proposed to refer to the ensemble of proteins constituting the extracellular matrix (core matrisome) as well as the proteins associated with the extracellular matrix. The neurovascular system differs in genomic status and function depending on brain region and cell types. The transcriptome data from specific tissue and/or specific cells in BCAS mice will contribute to matrisome data construction. And ideally, beyond the transcriptome data, it would also be necessary to assemble the dataset of proteomic/metabolomic/lipidomic of BCAS mouse brains to further our understanding of SIVD pathology due to cerebral small vessel diseases.

Since this BCAS model was first reported in 2004, more than 100 papers have been published using this model. BCAS mice show several features of human SIVD, and we could say that this BCAS model is one of the well-accepted mouse models to study the pathology of SIVD (or other small vessel diseases). Studies with this model have revealed multiple cellular and molecular mechanisms which cause brain damage and repair. Also, the model has been utilized to test the efficacy of candidate drugs and non-pharmacological interventions for white matter function and cognitive function under the conditions of cerebral hypoperfusion. Therefore, it is expected that the BCAS model will continue to be used to pursue the pathological mechanisms of vascular-related dementia and to find an effective therapeutic approach for this disease.